Chlamydia trachomatis (C. trachomatis or CT) is a causative agent of common sexually transmitted diseases, including venereal lymphogranuloma, various inflammatory pathologies of the male and female urogenital systems, and trachoma, a chronic disease that affects 500 million people and can lead to blindness. When not timely diagnosed and treated, CT-induced urethritis and cervicitis can led to chronic inflammations, e.g., vaginitis, salpingitis and pelvic inflammation which can result in sterility and extrauterine pregnancy. Furthermore, newborns from infected mothers can contract pulmonary and/or ocular infections during delivery.
The Chlamydiae
Chlamydiae are prokaryotes that exhibit morphologic and structural similarities to Gram negative bacteria including a trilaminar outer membrane that contains lipopolysaccharide and several membrane proteins. Chlamydiae differ from other bacteria by their morphology and a unique developmental cycle. Obligate intracellular parasites, Chlamydiae have a unique biphasic life cycle consisting of a metabolically inactive but infectious extracellular stage and a replicating but non-infectious intracellular stage. The replicative stage of the life-cycle takes place within a membrane-bound inclusion that sequesters the bacteria away from the cytoplasm of the infected host cell.
Many different strains of Chlamydiae have been isolated from mammals (including man) and birds. Strains can be distinguished on the basis of host range, virulence, pathogenesis, and antigenic composition. There is strong DNA sequence identity within each species, but surprisingly little between species, suggesting long-standing evolutionary separation.
A cryptic plasmid (extra-chromosomal DNA) of about 7501 bp and having unknown function is found in almost all CT isolates and is known, for example, in the LGV strain as “pLGV440.” The plasmid is not essential for CT survival, but is remarkable in that the sequence is highly conserved across isolates (e.g., see (Comanducci et al., 1990)).
Diagnostic Tests
Rapid and specific diagnostic tests are of utmost importance for successful intervention against CT. Diagnosis based on selective growth of the pathogenic bacteria has been the standard, but cell culturing is time-consuming. Many clinical isolates are difficult to grow in vitro. Because bacterial infection causes antibody production in the host, sera from patients suffering genital tract infections have also been used to diagnose CT infection. However, assays based on serological markers are non-quantitative and often difficult to interpret. For example, antibody titers can be undetectable in acute infections (a false-negative result), persist in uninfected individuals with a past history of infection (a false-positive result), yield a false-positive due to the presence of cross-reacting species (e.g., respiratory infection by different Chlamydia species), or not develop at all (a false-negative result) depending on other factors Black et al., 1991; Ngeow, 1996). For these reasons, serology alone is inadequate for the diagnosis of CT infections.
There have been attempts to remedy these inadequacies. For example, the Abbott Laboratories REALTIME™ CT/NG (2G28) and CT (1L31) products use Chlamydia cryptic plasmid polymerase chain reaction (PCR) primers and probes in a homogenous real-time format (Pabich et al., 2004). Jalal et al. (Jalal et al., 2006) developed a rotor-gene, real-time PCR assay for detecting, identifying and quantifying CT in a single reaction. Picket et al. (Pickett et al., 2005) were able to determine accurately the copy number of CT and C. pneumoniae (N16) also using real-time PCR.
New Challenges in CT Detection
PCR-based methods of CT detection have mined the advantages of the cryptic plasmid found in almost all CT strains. The plasmid is favored as a target for polynucleotide-based diagnosis of CT infection because per bacterium, there are approximately 4-10 copies of the plasmid (Jalal et al., 2006; Palmer and Falkow, 1986; Pickett et al., 2005; Tam et al., 1992).
However, a new variant of CT with a deletion in the plasmid has been detected in Sweden (“CTSW”), following an unexpected 25% decrease in CT infections that was noted in Halland county, Sweden (Ripa and Nilsson, 2006).
For the past decade, laboratories in Sweden have used nucleic acid amplification tests (NAAT) using the cryptic plasmid as the template for DNA amplification to diagnose CT infections.
From mid-September to October 2006, the county microbiology laboratory in Halmstad, Halland county, tested 1700 consecutive incoming specimens with a major outer membrane protein (MOMP)-specific PCR in parallel with Abbott's m2000 plasmid PCR. In 13% of all diagnosed CT cases in Halland county during this period, Ripa et al. found a variant strain that was only positive in MOMP tests. Clinical data indicates no difference from infections with the wild type strains. The strain seems to be spread throughout Sweden, although prevalence in these areas is still unknown.
The part of the plasmid from the variant strain was sequenced. Ripa et al. found a deletion of 377 base pairs in the target area for the CT NAAT tests manufactured by Abbott and Roche (nucleotides 654 to 1030 of Accession No. X06707; SEQ ID NO:4). Twelve variant strains have now been sequenced and found to have the same deletion (Ripa and Nilsson, 2006). Therefore, there exists a need in the art for new NAAT tests that are capable of detecting this variant strain as well as distinguishing this variant strain from other strains of CT.
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